Immunoassay Product And Process

ABSTRACT

The embodiments disclosed herein are directed to an apparatus useful in conducting detection of compounds on blotting membranes. The device is comprised of several layers including a porous support layer below the blotting membrane(s), a flow distributor above the blotting membrane(s) and optionally a well on the flow distributor to contain the liquid to the desired area and to allow for lower starting volumes of such liquid. Preferably, the flow distributor is a non-binding or low binding hydrophilic porous membrane such as a 0.22 micron membrane and the support layer is a grid or sintered porous material. The distributor and support are held together to form an envelope around the membrane(s). The use of a hinge, clips and other such devices is preferred in doing so.

This application claims priority of provisional application Ser. No.61/484,701 filed May 11, 2011, the disclosure of which is herebyincorporated by reference.

The embodiments disclosed herein relate to a device and process for thedetection and position of substances that are contained in a blottingmembrane. More particularly, it concerns a technique for applyingreagents, wash solutions and detection chemistries to a blottingmembrane to accomplish this detection quickly via the use of vacuum orpositive pressure.

BACKGROUND

The use of gel electrophoresis is currently the ubiquitous technique forthe separation of biological materials. Nonbiological materials can alsobe separated using gels or other chromatographic supports as well, butthe scope of effort with regard to biologicals is greater. Typicalapplications include separation of nucleic acid fragments of varioussizes either in the context of sequence determination; in the detectionof polymorphisms; or verification of sizes in other contexts. Alsofrequently conducted are separations of proteins, glycoproteins, proteinfragments and application of gel separations as verification ofhomogeneity or purity, identification of post translationalmodifications and confirmation of molecular weight.

In all of these procedures, mixed samples of biological entities areapplied to electrophoretic gels and the components are separated byapplication of an electric field across the gel. Regardless of themanner in which the gel is developed, the resulting pattern of migrationof the substances contained in the sample must be detected in somemanner.

To conduct this detection, typically the gel support is contacted with ablotting membrane to which the substances are transferred in the samepattern in which they appeared on the gel. The “spots” are thendetected, at a minimum, by blocking the membrane with a protein ordetergent solution to reduce non-specific binding (which otherwise leadsto a high level of noise and low level of detection). Typical blockingagents include casein, bovine serum albumin (BSA), non-fat dry milk(generally about 1-5%) in a Tris buffer saline solution with TWEEN®surfactant (TBS-T solution) or phosphate buffer saline solution withTWEEN® surfactant (PBS-T solution). The biological entity is thenincubated with an antibody specific for the antigen on the membrane. Themembrane is then extensively washed to remove any contaminants, unboundblocking proteins or antibodies and the like. The membrane is thentreated and incubated with a secondary enzyme-, radioisotope-,fluorfluor-, or biotin-conjugated antibody specific for the primaryantibody. The membrane is then extensively washed again to remove anyunbound secondary antibody. Then a detection reagent, generally achromogenic, chemiluminescent, fluorescent, radiological, orstreptavidin-labeled material, is applied which either binds to, or is asubstrate of the enzyme-conjugate. Lastly, the appropriate detectiondevice is used to determine the presence, absence, position, quantity,etc. of the biological entity. The last six steps generally take from3-6 hours to overnight depending on the speed of the reaction betweenthe selected reagents, the membrane and the biological entity. Theprocess requires multiple incubation periods of the membrane on arocking or other suitable mixing platform. It is a lengthy process thatmost researchers dislike and which consumes (wastes) a large volume ofreagents.

Some researchers have suggested the use of the capillary action of anabsorbent material such as filter paper placed below the membrane todraw the remaining fluids through the membrane and improve the speed ofthe process especially the washing steps.

U.S. Pat. No. 5,155,049 mentions a system called the Hybrid-Ease™hybridization chamber marketed by Hoefer Scientific Instruments. Thischamber is comprised two grids between which the membrane is sandwiched.The grid plates are snapped into position surrounding the membrane, andsyringes fitted into the open space created by the grids. One syringe isused to apply reagents and wash, and the other to withdraw excess. Thesystem requires large volumes of liquid in order to operate, iscumbersome to employ and is still quite time consuming. It also mentionsthat in some particular assays, such as ELISA assays, in small volumewells (such as 96 well microtiter plate), others have used vacuum todraw liquids through a membrane in a washing step. However, theydiscount this effort as it is only available in small volumeapplications and still is uncontrollable. They suggest instead that thebetter method is to use a manual press having the membrane on top of afilter paper and cover layer and then pressing the membrane sandwichbetween two plates to squeeze the liquid through the membrane and intothe paper.

In U.S. Ser. No. 60/732,994, filed Nov. 3, 2005 it is suggested that oneuse a device formed of several layers including a porous support layerbelow the one or more layers of blotting membrane, a flow distributorabove the blotting membrane(s) and a well on the flow distributor tocontain the liquid to the desired area and to allow for lower startingvolumes of such liquid. Preferably, the flow distributor is anon-binding or low binding porous membrane

In co-pending U.S. Ser. Nos. 11/582,727 and 11/582,599 filed on Oct. 18,2006, a device was disclosed in which a holder having a flow directorand a porous support could be fit into a manifold apparatus to processthe various fluids and detect the biological entity. The manifold has acover with or without a well and a central opening inline with a centralopening of the holder. While useful it has limitations that inhibit itsuniversal use and acceptance.

It is clear that a more efficient method for detection of the biologicalmaterials or entities on blotting membranes is required. The embodimentsdisclosed herein permit a more effective and efficient detection ofbiological entities in a blotting membrane.

SUMMARY

In accordance with certain embodiments, an immunodetection system andmethod are provided. The system comprises a manifold base adapted tocommunicate with a driving force such as vacuum. The manifold holds acarrier that supports a blot membrane holder. The blot membrane holderholds the blot membrane to which proteins can be bound by way ofelectrophoresis. The system and method enable users to quickly andefficiently prepare protein blots for detection such as bychemiluminescence.

In one embodiment, there is provided an apparatus useful in conductingthe method disclosed herein. The device is comprised of a blottingmembrane holder (also referred to as a blot holder, or simply as aholder) formed of a lower porous support layer and an upper flowdistributor. The two are held together by a method such as by a hinge,clips, elastic bands, adhesives, ball and socket, pins and recesses, orcooperatively engaging fasteners or other such means. The holder isopened and one or more blotting membranes are placed between the lowerand upper layers. The holder is then closed and placed into a carrierformed of an upper and lower plate, each with at least one opening thatallows fluid to pass through the top plate of the carrier, through theholder and membranes contained within it, and then through the lowerplate of the carrier. The carrier is then set into the manifoldapparatus to process the samples on the blotting membrane. Uponcompletion of the process, the carrier is removed from the manifold,opened, and the blot membrane holder is removed. The blot membraneholder is then opened and the blot membrane is extracted for downstreamdetection.

In another embodiment, the holder is formed of a material selected fromthe group consisting of plastic and paper, the holder has a top portionand a bottom portion, each top and bottom portion has an outer edge, atop surface and a bottom surface and a thickness between the top andbottom surfaces, at least one of the portions has a solid portion inwardof the outer edge, at least the top portion has an opening inward of theouter edge, a porous support formed on the top surface of the bottomportion and a flow distributor formed on a lower surface of the topportion which distributor covers the opening of the top portion whereinthe holder has a means for releasably securing the first and secondportions to each other when the top and bottom portions of the holderare aligned such that the bottom surface of the top portion and the topsurface of the bottom portion are aligned and arranged adjacent eachother to bring the top and bottom portions together.

In another embodiment, the holder is formed so that the top and bottomportions each has a perforation along one side and the perforation isaligned on the top and bottom portions, and the perforations are easilytorn to open the holder to retrieve the blot membrane

In one embodiment, the carrier has an outer perimeter wall extendingupwardly from the opening of the top plate of the carrier to form a wellto hold reagents and washing fluids.

In a further embodiment, a carrier has one or more level indicators toindicate whether the top surface of the carrier is level in thehorizontal plane along at least its length and preferably along itslength and width. The level also could be on the manifold

In another embodiment, the level indicator is a single 360 degree bubbleindicator.

In a further embodiment, the level indicators are at least two linelevel bubble indicators with at least one arranged along the length ofthe top surface of the top plate and at least one arranged along thewidth of the top surface of the top plate.

In a further embodiment, the carrier inner surface(s) contains a featureto stretch the one or more blotting membranes in the holder so as toavoid the formation of wrinkles in the membrane and make the surface ofthe membrane flat or planar as possible.

In another embodiment, the carrier has a stretching feature formed of aflange seal located on at least one of the inner surfaces of the top orbottom plates. Preferably it has a flange seal formed on at least theadjacent inner surfaces of the bottom plate. More preferably, the flangeseals are located at a width and length that is greater than the openingof the holder but less than the width and length of the outer edge ofthe holder.

In another embodiment, the carrier has a stretching feature consistingof a compressible or flexible member positioned on one half of thecarrier with an opposing raised rigid feature on the other half of thecarrier.

In one embodiment, the manifold is subdivided into two or more subwellsto run parallel blotting membranes or subparts of one blotting membrane,each membrane is typically processed with at least one differentreagent.

In another embodiment, the carrier is subdivided into two or moresubwells with the holder also subdivided into corresponding subwells.

In another embodiment, the carrier subwells are individually sealed andaligned with the corresponding holder subwells so as to prevent fluidmigration between wells.

In another embodiment, the manifold has leveling feet that when used inconjunction with one or more level indicators of one embodiment of thecarrier allow the manifold and carrier to be leveled in a horizontalplane.

In one embodiment, the manifold has a series of feet that are capable ofbeing adjusted in a vertical direction so as to level the manifold inrelation to the level indicator(s) on the carrier when the carrier isattached to the manifold.

In further embodiment, the series of feet have a screw that fits into athreaded portion of the manifold bottom to allow the feet to beindividually raised or lowered in a vertical direction.

In another embodiment, the manifold is subdivided into two or moresubwells to run parallel carriers with different blotting membranes orsubparts of one blotting membrane, each membrane is typically processedwith at least one different reagent.

In another embodiment, the carrier top plate opening has a lidpositioned onto the upper edge of the opening to minimize evaporationduring long term, i.e. overnight incubation.

In another embodiment, the lid has a resealable relation with the well.

In another embodiment, the lid is light blocking such as being made froman opaque material.

In another embodiment, the opening on the carrier bottom plate has acover to prevent fluid leakage.

In another embodiment, the cover is releasably sealed.

In another embodiment, the cover has a valve feature.

In another embodiment, the valve is of a duck bill or umbrellaconfiguration.

In another embodiment, the valve is operational by a pressuredifferential and closed when no pressure differential is present acrossthe valve.

In another embodiment, a rapid, efficient and convenient method todetect one or more biological entities on a blotting membrane isprovided. The detection can relate to the position, nature or amount ofthe biological substance on a membrane. The method involves a pressureassisted regiment, selected from positive pressure or a vacuum for thesupply and removal of reagents to and from the blotting membrane andpermits washing of the contaminants from substances embedded in themembrane that are to be detected using very low volumes of liquid andreagents. This method enables completion of the blocking, washing andantibody binding steps in about 30-45 minutes without compromising blotquality. One simply takes a holder, opens it and places the blottingmembrane(s) on one of the surfaces such that the lower surface of theblotting membrane is adjacent the porous support and the upper surfaceof the blotting membrane is adjacent the flow distributor when thedevice is closed around the membrane(s). The device is placed on or in amanifold having a pressure or vacuum supply and the process iscommenced.

It is another object of the embodiments disclosed herein to provide anapparatus for conducting pressure or vacuum assisted immunoassays of oneor more blots comprising a vacuum manifold, a carrier designed to fit onthe manifold and a holder designed to fit within the carrier forprocessing the blots and a mean of collecting one or more of theantibodies.

It is a further object of the embodiments disclosed herein to provide aprocess for conducting vacuum assisted immunoassays on one or moremembranes comprising the steps of:

-   -   a. providing a vacuum manifold, a carrier for holding a holder,        the carrier being formed of a top plate and a bottom plate each        with at least one opening formed through its thickness to allow        fluid to flow through it, a holder for the one or more blotting        membranes, the holder being capable of being positioned between        the top and bottom plates of the carrier and being in fluid        communication with the at least one opening of each plate of the        carrier, the holder being formed of a porous support and a flow        distributor which are held together, one or more membranes        containing one or more biological entities to be assayed, the        membrane(s) being placed on the porous support, a flow        distributor being on top of the membrane and the holder being        placed between the top and bottom plates of the carrier such        that the flow distributor is adjacent the bottom or inner        surface of the top plate and the porous support is adjacent the        inner of top surface of the bottom plate,    -   b. adding one or more reagents to the at least one opening of        the carrier top plate and applying a vacuum to pull the reagents        into the membrane through the opening in the top plate, the flow        distributor and porous support of the holder and the opening of        the bottom plate, and    -   c. adding one or more washing agents to the one or more wells        and applying a vacuum to pull the washing agents and any unbound        reagents through the top plate opening, flow distributor,        membrane and porous support and opening of the bottom plate of        the carrier and into the vacuum manifold and    -   d. repeating steps (b and c) one or more additional times as        desired or required.

In certain embodiments, disclosed is a device for conductingimmunoassays comprising a holder formed of a material selected from thegroup consisting of plastic and paper, the holder having a top portionand a bottom portion, each top and bottom portion having an outer edge,a top surface and a bottom surface and a thickness between the top andbottom surfaces, at least one of the portions has a solid portion inwardof the outer edge, at least the top portion has an opening inward of theouter edge, a porous support formed on the top surface of the bottomportion and a flow distributor formed on a lower surface of the topportion which distributor covers the opening of the top portion, whereinthe holder has a means for releasably securing the first and secondportions to each other when the top and bottom portions of the holderare aligned such that the bottom surface of the top portion and the topsurface of the bottom portion are aligned and arranged adjacent eachother to bring the top and bottom portions together.

In certain embodiments, the means for releasably securing may comprise asealing material formed outwardly and circumscribing the porous supportopening. The top and bottom portion of the holder may be made of onepiece of material and the holder may have a fold running a width of theholder to form a first portion and second portion. The fold may be ahinge.

In certain embodiments, the flow distributor may have a lower and anupper surface and the upper surface of the flow distributor may beattached to the bottom surface of the top portion of the holder suchthat the thickness of the first portion forms one or more wells on theupper surface of the flow distributor. The opening of the top portion iscentrally located within the outer edge of the top portion.

In certain embodiments, the device for conducting immunoassays maycomprise a holder and a carrier for the holder, the holder being formedof a material selected from the group consisting of plastic and paper,the holder may have a top portion and a bottom portion, each top andbottom portion may have an outer edge, a top surface and a bottomsurface and a thickness between the top and bottom surfaces, at leastone of the portions may have a solid portion inward of the outer edge,at least the top portion may have an opening inward of the outer edge, aporous support formed on the top surface of the bottom portion and aflow distributor formed on a lower surface of the top portion whichdistributor covers the opening of the top portion wherein the holder mayhave a means for releasably securing the first and second portions toeach other when the top and bottom portions of the holder are alignedsuch that the bottom surface of the top portion and the top surface ofthe bottom portion are aligned and arranged adjacent each other to bringthe top and bottom portions together.

In certain embodiments, the device also may comprise a carrier for theholder comprised of a top plate and a bottom plate, each having a widthand a length, a top and bottom surface and a thickness between the topand bottom surfaces, an outer edge and at least one opening, the platesbeing of a length and width greater than the length and width of theholder, the top plate of the carrier having an opening substantiallyequal in width and length to the opening of the top portion of theholder, the bottom surface of the top plate and the top surface of thebottom plate each having one or more seals in alignment with each otherwhen the two plates are adjacent each other and the seals being arrangedon each surface at a width and length greater than that of the openingof the top plate, but less than the outer dimensions of the holder, atleast one of the plates has a seal formed adjacent the outer edge of theplate and outward of the seal of that plate, and a means for releasablyholding the top and bottom plate together.

In certain embodiments, the seals in the bottom surface of the top plateand the top surface of the bottom plate may be flange seals.

In certain embodiments, there may be at least one level indicatingdevice attached to the upper surface of the top plate, and it mayinclude a 360 degree indicator. There may be two level indicators, onealong the length and the other along the width of the top surface of thetop plate.

In certain embodiments, disclosed is a device for conducting vacuumassisted immunoassays comprising a vacuum manifold and a holder formedof plastic or paper, the holder has a fold running a width of the holderto form a first portion and second portion, each portion has an openingthat are aligned when the holder is folded closed along the fold, aporous support covers a first opening and a flow distributor covers thesecond opening wherein the holder has a means for releasably securingthe first and second portions to each other when the holder is foldedclosed along the fold to bring the first and second portions together.The flow distributor may be a membrane.

In certain embodiments, disclose is a device for conducting immunoassayscomprising a vacuum manifold having a base, the base having an uppersupport surface for supporting one or more carriers that contain aholder to be processed and a drain below the support, the upper surfacecontaining one or more central openings extending through it and the oneor more central openings are in alignment with the one or more carriers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exploded view of a carrier in accordance with certainembodiments;

FIG. 2 is a perspective view of a carrier in an open position inaccordance with certain embodiments;

FIG. 3 is a perspective view of a carrier in a closed position inaccordance with certain embodiments;

FIG. 4 is a perspective view, shown in cross-section, of a carrier inaccordance with certain embodiments;

FIG. 4A is a perspective view, shown in cross-section, of a carrier withan umbrellas valve in accordance with certain embodiments;

FIG. 5 is a perspective view of a blot membrane holder, including ablotting membrane, in accordance with certain embodiments;

FIG. 5A is a perspective view of a blot membrane holder, showing a flowdistributor, in accordance with certain embodiments;

FIG. 5B is a perspective view, viewed from the underside, of a blotmembrane holder, in accordance with certain embodiments;

FIG. 6 is a perspective view of a carrier in an open position, includingthe holder of FIG. 5, in accordance with certain embodiments;

FIG. 7 is an exploded perspective view of the underside of the carrierin accordance with certain embodiments;

FIG. 8 is a perspective view of the underside of the carrier with thecover in place, in accordance with certain embodiments;

FIG. 9 is a perspective view of a manifold in accordance with certainembodiments;

FIG. 9A is a perspective view of a manifold with a collection vessel inaccordance with certain embodiments;

FIG. 10 is an exploded perspective view of a manifold, includingcarriers, in accordance with certain embodiments; and

FIG. 10A is a perspective view of a manifold, holder and carrier inaccordance with certain embodiments.

DETAILED DESCRIPTION

Turning first to FIG. 1, there is shown a carrier 10 in accordance withcertain embodiments. In the embodiment shown, the carrier 10 includes alid 11, a carrier top plate 12, a carrier bottom plate 13, a cover 14,and a bubble level 15 on the carrier top plate 12. In accordance withcertain embodiments, the carrier 10 serves to hold the blot holder flatand to deliver reagents such as antibodies, wash buffer, etc. to theblot holder. In accordance with certain embodiments, the carrier 10 is astandalone device, separate from a manifold base (discussed below),which allows the user to setup multiple blots at the same time. Thus,each carrier 10 can be selectively loaded with blot holders containingblots, their respective antibodies added, and then incubated.

FIG. 2 shows an embodiment of the carrier 10 in an open position, withthe underside of the top plate 12 shown to the left in the drawing andthe top side of the bottom plate 13 shown to the right. In theembodiment shown, the top plate 12 and bottom plate 13 are hingedlyconnected at 16. As shown in this embodiment the hinge is a “live” hingethat bonds the two portions together. Alternatively, the hinge could bemade separately and attached using adhesives, heat bonds or mechanicalfasteners. Other embodiments use no hinge (not shown) and use clips,elastic bands or cooperatively engaging fasteners such as a slot anddetent, friction fit pin or the like form on or in the respective topand bottom portions to hold them together during use. Other comparablemeans will be obvious to one of skill in the art and it is meant toinclude them as well.

In accordance with certain embodiments, the carrier top plate 12 mayeither have one or more wells 22, which can be used for holding and/ordelivering washing fluids and reagents during use. The well(s) 22 mayeither be formed as part of the top surface of the top carrier plate 12with upstanding walls 22 a, 22 b, 22 c and 22 d (FIG. 1), or as aseparate piece which is simply attached or placed on top of the carriertop plate 12. The lid 11 can be placed on the carrier 10 to cover thewell 22 and prevent antibodies and other fluids from evaporating duringextended incubation periods, particularly during overnight incubation.

The carrier bottom plate 13 includes a porous support 4 that may be asimple screen, a grid, a flow directing grid or a sintered porousstructure such as a POREX® membrane or a coarse or large poredmicroporous filter, such as a woven or non-woven paper, a polypropyleneor polyethylene fabric, a glass mat or paper, or a 1-10 micronmicroporous filter. Such supports can be made of polymer, glass, ceramicor metal materials including but not limited to metals, such asstainless steel or steel alloy, aluminum and the like, and polymers suchas polyethylene, polypropylene, polysulfone, polyethersulfones,styrenes, nylons and the like. FIG. 2 shows a porous support 4 in theform of a flow directing grid including a series of grooves 72 andopenings 74. The openings 74 are inwardly positioned from the perimeterof the porous support 4. The openings 74 are in fluid communication withthe grooves 72 so that fluid is collected in the grooves 72 and directedthrough the openings 74. The grooves 72 collect and deliver the spentfluid to the openings 74 which direct the fluid to a waste chamber orcollection tray (not shown). If the researcher wishes to collect one ormore of the fluids, then a collection tray can be appropriatelypositioned to do so. Those skilled in the art will appreciate that otherpatterns of grooves or openings may be used, as the desired outcome isto direct the spent fluids to an opening or a series of openings thatdirect the spent fluids to a predetermined destination.

The outer edges of the support 4 and the carrier top plate 12 may bemade of the same materials as the support 4. When an integral hinge isused, it must be made of a flexible material such as polyethylene,polypropylene, an elastomer or one of the impact modified materials suchas ABS, K-resin and the like. When a separate hinge, clips, elasticbands, adhesive film or other securing means are used they may be madeof metal, plastic or elastomers as desired.

In accordance with certain embodiments, a first flange seal 30A ispositioned on the underside of the top carrier plate 12 as shown, andcooperatively with a second flange seal 30B positioned about the poroussupport 4 on the carrier bottom plate 13, maintains a fluid tight systemwhen the top and bottom plates are in the closed position with the blotholder engaged. Thus, the flange seals 30A and 30B are positioned sothat when the top carrier plate 12 is rotated with respect to the bottomcarrier plate 13 to a closed position, the two seals register or alignwith one another to seal the carrier. In certain embodiments, eachflange seal 30A, 30B is preferably made of a flexible elastomer such assilicone, or thermoplastic elastomer (TPE) and is V-shaped, with one legof the V being short than the other. The long end of each V-shapedflange seal seats in a respective recess of the top and bottom carrierplates (FIG. 4). The short leg of each V-shaped seal is a raised, easilydeformable feature. In the carrier closed and sealed position, theshorter legs of the V on the top carrier plate will contact the topsurface of the blot holder. The shorter leg of the V on the bottomcarrier plate will contact the bottom of the blot holder and deflect asbest seen in FIG. 4. The angled geometry of the seal exerts a force topull or stretch the holder 50 when deflected, which “drums” the membraneand keeps it flat. A flat membrane avoids deformities that can createpeaks and valleys on the membrane surface which pools the antibodyvolume, which in turn leads to areas of the blot membrane having more orless exposure to the antibodies, resulting in poor and inconsistentresults. This seal prevents leaking and enables long term (e.g.,overnight) incubation. The design of the seal also allows for a lowforce sealing mechanism and low clamp force required for the carrier 10.This helps to maintain flatness of the holder and the blot membrane. Areleasable latch 35 or other locking mechanism locks the carrier in theclosed position (FIG. 3). In accordance with certain embodiments, asingle flange seal can be used, preferably the flange seal 30B for thebottom plate. The bottom flange seal 30B functions to stretch the blotholder. Once stretched, the interface is designed so that the carriertop and bottom pinch the blot holder in place. Outer flange seals 300A,300B are also shown in FIG. 4.

FIGS. 5, 5A and 5B show a holder 2 in accordance with certainembodiments. The holder 2 includes a frame 40 having a perimeter edgeconfigured to support a protein blotting membrane 45. In certainembodiments, the frame 40 is constructed of plastic or paper. In certainembodiments, the material of the frame 40 can be bioresin, cardboard,HIPS, paper, or any other suitable rigid material that can support themembrane and keep it flat. In accordance with certain embodiments, aflow distributor 46, such as a GVPP membrane, is laminated or otherwiseattached to the frame, and over which over the blotting membrane 45 canbe positioned. The flow distributor 46 is attached to the frame and theblot membrane is sandwiched between the flow distributor 46 and theporous support 47 when the blot holder is closed. The porous support 47on the blot holder is supported and in contact with the porous support 4of the carrier bottom plate. The flow distributor 46 functions to evenlydistribute the blocking solution and the antibodies over the blotmembrane. It also regulates flow through of these solutions when vacuumis applied. The holder 2 also includes a porous support 47, such as apiece of polypropylene non-woven mesh bound to the frame along one edge,allowing easy access for the user to add the blot membrane, and whichsupports the blot membrane and protects it from damage under vacuumforce. The support 47 is porous to allow free flow of liquids throughthe blot holder 2. The holder 2 is shown properly positioned in thecarrier 10 in FIG. 6.

In certain embodiments, the holder 2 can be made by laminating apressure sensitive adhesive to the back of a polypropylene mesh suchthat after adding the blot membrane to the holder, the user removes abacking strip to expose the pressure sensitive adhesive and then sealsthe mesh to the flow distributor (e.g., to the GVPP membrane) to createa sealed envelope. To remove the blotting membrane, perforations andzippers can be included to tear open the envelope.

In certain embodiments, a thin patapar paper, wax paper or other thinsheet of paper material is laminated to one side of a polypropylenemesh. When the holder is wet with water (as is required in the process),the wet paper backing sticks to the GVPP membrane, creating a temporaryseal. After the process is complete, the blot membrane is easy to removewithout having to tear anything or manage sticky adhesives. The holderis then disposed of, as after the paper backing dries, it curls and theholder becomes unusable.

FIGS. 7 and 8 illustrate the underside of the bottom carrier plate 13.Visible in FIG. 7 are the plurality of openings 74 in the porous support4. Cover 14 is used to cover the openings to prevent leaking ofantibodies during incubation, particularly when incubating for longperiods of time, such as overnight, as seen in FIG. 8. In accordancewith certain embodiments, the openings 74 are surrounded by an annularraised ring 49, and the cover 14 has a corresponding annular slot 60(FIG. 4) configured to mate with the annular ring 49 and thus the covercan be pressed onto the ring to secure it to the carrier plate 13.

Alternatively, the cover 14 can be eliminated, and an umbrella valve 144can be used to retain the liquid during incubation, as shown in FIG. 4A.When the vacuum is turned on, the valve opens and the liquid drains.Preferably the valve is made of EPDM (ethylene propylene diene monomer)due to its stiffness and ability to snap closed quickly when the vacuumis turned off. This helps to prevent backpressure and air trapping,which (with the silicone valves) was causing the blot holder to bow andlead to adverse antibody pooling. Antibody pooling (non uniformcoverage) leads to a non-uniform signal on the final blot readout-areasnear the trapped air will have a weak signal (faint) and areas withpooled antibody will have a strong signal (bold).

As shown in FIGS. 9 and 10, in accordance with certain embodiments,manifold 8 is a vacuum manifold that can be attached to a suitablesource of vacuum (not shown). The manifold 8 may include a wastecollection device (not shown), such as a receptacle, positioned in themanifold (not shown) to collect the liquid pulled through the carrier10.

In accordance with certain embodiments, the manifold 8 has a base 42,having a drain and support surface 44 on which one or more carriers 10is placed. The support surface 44 includes one or more wells or carrierreceiving regions 55A, 55B (two shown in FIG. 9) that may include aplurality of upstanding ribs 56 that receive the underside of thecarrier bottom plate 13 to hold the carrier 10 in a stable manner in themanifold 8. Also shown are optional respective controls 62 for managingand monitoring the manifold 8 and the process. The device can be usedwith automated liquid handlers and the like if desired. Where themanifold 8 is subdivided into one or more wells or carrier receivingregions, the manifold can process more than one holder to run parallelcarriers with different blotting membranes or subparts of one blottingmembrane, each membrane is typically processed with at least onedifferent reagent. The manifold 8 can have a common pressure source oreach station carrier receiving region can be pressure controlledindividually.

In another embodiment, the manifold has leveling feet that when used inconjunction with one or more level indicators of one embodiment of thecarrier allow the manifold and carrier to be leveled in a horizontalplane. In one embodiment, the manifold has a series of feet that arecapable of being adjusted in a vertical direction so as to level themanifold in relation to the level indicator(s) on the carrier when thecarrier is attached to the manifold. In further embodiment, the seriesof feet have a screw that fits into a threaded portion of the manifoldbottom to allow the feet to be individually raised or lowered in avertical direction.

Various methods may be used in the embodiments disclosed herein. The keyfactor being that they all rely on a vacuum or positive pressure drivenfiltration of the liquids to access the large inner surface area of themembrane allowing 3-D interaction of all the molecules throughout thedepth rather than only 2-D interaction at the surface as has occurred inthe past. Where positive pressure is used, the base could be a closedchamber that could contain the carrier, and the lid 11 would become apressure manifold to apply pressure to the well 22 of the carrier.

The simplest method is to use the embodiments disclosed herein toconduct one or more of washing cycles. Typically each washing cycle iscomprised of one or more washing steps. Generally, 2-5 steps are usedper cycle.

Another method is to use the embodiments disclosed herein in each stepin which liquid needs to be moved through the blotting membrane such asafter incubation of the antibodies or in the washing steps.

In all of these processes, any driving force suitable to move theliquid(s) through the device and into the manifold can be used. This canvary depending upon the membranes selected for blotting and the manifoldused, the desired speed of the filtration and the supply of vacuum orpositive pressure available to the researcher.

Generally, the vacuum available may vary between 100 and 760 mm Hg (133millibars and 1013 millibars). The use of valves, pressure restrictorsand the like may also be used to keep the vacuum within the allowedranges for the membranes used. A preferred vacuum manifold of oneembodiment uses of a vacuum of about 100 mm Hg. Other suitable vacuummanifolds include but are not limited to the MULTISCREEN™ andMULTISCREEN™_(HTS) vacuum manifolds available from Millipore Corporationof Billerica, Mass.

Generally the positive pressure is supplied by an air line at pressuresranging from about 2 psi to about 15 psi. The use of valves, pressurerestrictors and the like may also be used to keep the pressure withinthe allowed ranges for the membranes used. Such pressure systems includebut are not limited to Amicon® stirred cell devices available fromMillipore Corporation of Billerica, Mass. and positive pressurefiltration units available from Caliper Life Sciences of Hopkinton,Mass.

To use a device according to the embodiments disclosed herein, onesimply takes a holder, opens it and places the blotting membrane(s) onone of the surfaces such that the lower surface of the blotting membraneis adjacent the porous support and the upper surface of the blottingmembrane is adjacent the flow distributor when the device is closedaround the membrane(s) so as to have no air bubbles between the blot andthe flow distributor. Bubbles between these two surfaces can cause areasof no flow. The blot holder is then placed inside the carrier which isthen securely closed with the latch mechanism. The device is placed onor in a manifold having a pressure supply (vacuum or positive pressure).Preferably the blotting membrane(s) has been prewet. The pressure(vacuum or positive pressure) is turned on and a liquid, such as a washliquid or a reagent, is placed in the well of a carrier. The pressurecontinues until the liquid has been moved through the device andmembrane(s). Then the pressure is turned off.

When more than one blotting membrane is used, they can be arranged inseries on top of each other and sufficient liquid containing the samedesired reagents can be easily moved through the multiple layers in oneprocess step. Generally when more than one layer is used it is preferredthat one use between 2 and 10 layers, preferably between 2 and 5 layersat a time.

The liquid can either be added with the pressure supply being off or thesupply being turned on only briefly so as to get the liquid into themembrane(s) and is allowed to incubate (such as may be required with theprimary or secondary antibodies). The pressure is then turned on toremove the liquid and/or replace it with another used sequentially.Preferably, during washes, the vacuum is left on and remaining washesare added sequentially.

Optionally, if one wishes, one can place a collection vessel 70 belowthe device (FIG. 9A), preferably in the manifold itself or downstream,such as an antibody collection tray. It can then be used to collect oneor more unbound reagents that may be expensive and which can becollected and recycled for use in future assays. The vessel can also besubdivided into multiple chambers that are in alignment and fluidcommunication with the respective portion of the blotting membrane.

Additionally or alternatively, one can place in the downstream flow pathbelow the holder an absorbent matrix that is capable of reversiblybinding one or more unbound reagents that are expensive. The matrix ispreferably in the form of a monolith, such as a pad, a plug or a papersheet, that is positioned so that all the liquid passing through theblotting membrane and holder passes through the matrix. It can theneither be removed and the reagent eluted or if desired, it can have thebound reagents eluted in situ after completion of the testing of theblotting membrane.

Other processes may also be used with the device of the embodimentsdisclosed herein.

The membrane contains, in its interstices, one or more substances to bedetected. Generally these substances are present in the intersticeseither by virtue of having been blotted from a solid support forelectrophoresis or chromatography or by direct application, usually todetect the presence, absence, or amount of a particular type of materialsuch as an antibody or specific protein—i.e. a Dot-Blot type assay asdescribed above. The definition of the membrane is not limited, however,to these instances, but applies to any case wherein a membrane containsin its interstices one or more substances to be detected. Included inthe types of membranes envisioned for use in the embodiments disclosedherein are membranes commonly used to blot electrophoresis gels such asnitrocellulose; nylon; or various other polymeric membranes, such aspolyvinylidene fluoride (PVDF), sold as IMMOBILON™ membranes byMillipore Corporation of Billerica, Mass.

A variety of materials can be used to replicate the results ofelectrophoresis gels performed on various samples as is understood inthe art. Most commonly, the samples contain biological substances suchas individual proteins, antibodies, nucleic acids, oligonucleotides,complex carbohydrates, and the like, but the application of thetechnique is not limited to these substances. The technique isapplicable to any membrane containing within it a substance to bedetected regardless of the chemical composition of the membrane or ofthe target substances.

When membranes which represent replicas of electrophoretic results areemployed, the transfer of the substances to be detected from the gel tothe membrane can be conducted by utilizing membranes containing transferbuffer, by electroelution, or by dry blotting of the gels. Techniquesfor these transfers are well understood in the art, and do notconstitute part of the embodiments disclosed herein.

The liquid to be supplied may contain detecting reagents or may simplybe provided as a wash. The nature of the detecting reagent depends, ofcourse, on the substance to be detected. Typically, proteins aredetected by immunological reactions between antigen and antibody orimmunoreactive portions thereof; typically the presence of nucleic acidfragments is detected by suitable oligonucleotide probes. The detectingsubstances responsible for the immediate or specific reaction with thesubstance to be detected may be further supplemented, if needed, withlabel and a multiplicity of applications of the detecting reagents maybe needed—e.g., a protocol may include detection of an antigen bysupplying an antibody labeled with an enzyme, e.g., commonly,horseradish peroxidase, and then this binding is detected by means ofsupplying substrate for this enzyme. In application of reagent, it ispossible, though not preferred, to use only a positively pressed donormatrix to expose this component of the membrane for a defined period.

It is most convenient to conduct the method of the embodiments disclosedherein at room temperature, but elevated and lower temperatures can alsobe used. This can be effected by heating the device, its surroundingenvironment (as in a heat box or cooling box) or the liquids used in thesystem.

Blots can be sequentially analyzed with multiple antibodies or probes inthe present device and process by stripping the previously boundantibodies from the blot followed by subsequent incubations withantibodies or other probes specific other target proteins. The strippingprocess disrupts the antigen-antibody bonds and dissolves the antibodiesin the surrounding buffer. This is usually achieved by a combination ofdetergent and heat or by exposure to either high or low pH. The device,in combination with the flow distributor, enables the stripping of blotsusing the high or low pH method. The subsequent reprobing of blotseither directly (e.g., using the same flow distributor used forstriping) or subsequently after storage, would use the same protocol asthe initial probing. Suitable kits for strip blotting are available fromChemicon International, Inc under the brand names of ReBIot™ Plus kit(catalogue #2500), ReBlot Plus-Mild solution (catalogue #2502) andReBlot Plus-Strong solution (catalogue #2504).

In standard western blotting, the antigen or target is transferred to amembrane support and probed with a suitable probe such as an antibody,protein (e.g., Protein A) or lectin (proteins or glycoproteins whichbinding to carbohydrate moieties). In some applications, a reverseformat (e.g., reverse array) is used, wherein the antibody or otherprobes are spotted onto a membrane or other support (typically in anarray format) and the antigen or target is presented to the immobilizedantibodies on the array. Visualization of a target-probe binding eventcan be achieved by labeling of the antigens or targets or by using asecondary antibody specific for the target. Reverse arrays often employmixtures of targets, for example lysates labeled with differentfluorescent colors to enable parallel processing. Reverse assays canalso be performed with the embodiments disclosed herein.

1. A device for conducting immunoassays comprising a holder formed of amaterial selected from the group consisting of plastic and paper, theholder having a top portion and a bottom portion, each top and bottomportion having an outer edge, a top surface and a bottom surface and athickness between the top and bottom surfaces, at least one of theportions has a solid portion inward of the outer edge, at least the topportion has an opening inward of the outer edge, a porous support formedon the top surface of the bottom portion and a flow distributor formedon a lower surface of the top portion which distributor covers theopening of the top portion, wherein the first and second portions arereleasably secured to each other when the top and bottom portions of theholder are aligned such that the bottom surface of the top portion andthe top surface of the bottom portion are aligned and arranged adjacenteach other to bring the top and bottom portions together.
 2. The deviceof claim 1, comprising a sealing material formed outwardly andcircumscribing the porous support opening to releasably secure saidfirst and second portions to each other.
 3. The device of claim 1wherein the top and bottom portion of the holder are made of one pieceof material and the holder has a fold running a width of the holder toform a first portion and second portion.
 4. The device of claim 1wherein the fold is a hinge.
 5. The device of claim 1 wherein the flowdistributor has a lower and an upper surface and the upper surface ofthe flow distributor is attached to the bottom surface of the topportion of the holder such that the thickness of the first portion formsone or more wells on the upper surface of the flow distributor.
 6. Thedevice of claim 1 wherein the opening of the top portion is centrallylocated within the outer edge of the top portion.
 7. A device forconducting immunoassays comprising a holder and a carrier for theholder, a. the holder being formed of a material selected from the groupconsisting of plastic and paper, the holder has a top portion and abottom portion, each top and bottom portion has an outer edge, a topsurface and a bottom surface and a thickness between the top and bottomsurfaces, at least one of the portions has a solid portion inward of theouter edge, at least the top portion has an opening inward of the outeredge, a porous support formed on the top surface of the bottom portionand a flow distributor formed on a lower surface of the top portionwhich distributor covers the opening of the top portion, wherein thefirst and second portions are releasably secured to each other when thetop and bottom portions of the holder are aligned such that the bottomsurface of the top portion and the top surface of the bottom portion arealigned and arranged adjacent each other to bring the top and bottomportions together; and b. a carrier for the holder comprised of a topplate and a bottom plate, each having a width and a length, a top andbottom surface and a thickness between the top and bottom surfaces, anouter edge and at least one opening, the plates being of a length andwidth greater than the length and width of the holder, the top plate ofthe carrier having an opening substantially equal in width and length tothe opening of the top portion of the holder, the bottom surface of thetop plate and the top surface of the bottom plate each having one ormore seals in alignment with each other when the two plates are adjacenteach other and the seals being arranged on each surface at a width andlength greater than that of the opening of the top plate, but less thanthe outer dimensions of the holder, at least one of the plates has aseal formed adjacent the outer edge of the plate and outward of the sealof that plate.
 8. The device of claim 7, further comprising means forreleasably holding said top and bottom plates together.
 9. The device ofclaim 7, wherein the seal in the top surface of the bottom plate areflange seals.
 10. The device of claim 7 further comprising at least onelevel indicating device attached to the upper surface of the top plate.11. The device of claim 10 wherein the level indicator is a 360 degreeindicator.
 12. A device of claim 7 wherein there are two levelindicators, one along the length and the other along the width of thetop surface of the top plate.
 13. A device for conducting vacuumassisted immunoassays comprising a vacuum manifold and a holder formedof plastic or paper, the holder has a fold running a width of the holderto form a first portion and second portion, each portion has an openingthat are aligned when the holder is folded closed along the fold, aporous support covers a first opening and a flow distributor covers thesecond opening wherein the first and second portions are releasablysecured to each other when the holder is folded closed along the fold tobring the first and second portions together.
 14. The device of claim 1wherein the flow distributor is a membrane.
 15. The device of claim 7wherein the flow distributor is a membrane.
 16. A device for conductingimmunoassays comprising a vacuum manifold having a base, the base havingan upper support surface for supporting one or more carriers thatcontain a holder to be processed and a drain below the support, theupper surface containing one or more central openings extending throughit and the one or more central openings are in alignment with the one ormore carriers.
 17. The device of claim 16 wherein the base has multiplestations to position multiple carriers.
 18. The device of claim 16wherein the base has multiple stations to position multiple carriers andeach carrier in each station is subdivided into two or more wells. 19.The device of claim 16 further comprising the base has one or morecontrols for managing and monitoring the manifold and its operation. 20.The device of claim 16 the base has multiple stations and a commonpressure source for each station.
 21. The device of claim 16 wherein thebase has multiple stations, the base has a common pressure source foreach station and each station is capable of being controlledindividually.
 22. The device of claim 16 wherein the holder is formed oflower porous support and an upper flow distributor and the holder iscapable of holding one or more membranes between the lower support andupper flow distributor, the holder being inserted onto the upper supportof the base and the cover retains the holder(s) in place on the base.23. The device of claim 1 wherein the carrier(s) have one or more wellsattached to the upper surface of the top plate.
 24. The device of claim1, wherein the carrier top plate has an opening and a lid positionedover the opening.
 25. The device of claim 24, wherein said lid is madeof a light blocking material.
 26. The device of claim 1, wherein saidcarrier bottom plate has an opening and a cover releasably sealed tosaid opening.
 27. The device of claim 1, wherein said carrier bottomplate has an opening and a valve.
 28. The device of claim 27, whereinsaid valve is of a duck bill or umbrella configuration.
 29. The deviceof claim 27, wherein said valve is operational by a pressuredifferential and is closed when no pressure differential is presentacross the valve.